McDonald J P, Rothstein R
Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Genetics. 1994 Jun;137(2):393-405. doi: 10.1093/genetics/137.2.393.
A direct repeat recombination assay between SUP4 heteroalleles detects unrepaired heteroduplex DNA (hDNA) as sectored colonies. The frequency of unrepaired heteroduplex is dependent on the mismatch and is highest in a construct that generates C:C or G:G mispairs and lowest in one that generates T:G or C:A mispairs. In addition, unrepaired hDNA increases for all mismatches tested in pms1 mismatch repair-deficient strains. These results support the notion that hDNA is formed across the SUP4 repeats during the recombination event and is then subject to mismatch repair. The effects of various repair and recombination defective mutations on this assay were examined. Unrepaired heteroduplex increases significantly only in rad52 mutant strains. In addition, direct repeat recombination is reduced 2-fold in rad52 mutant strains, while in rad51, rad54, rad55 and rad57 mutants direct repeat recombination is increased 3-4-fold. Mutations in the excision repair gene, RAD1, do not affect the frequency of direct repeat recombination. However, the level of unrepaired heteroduplex is slightly decreased in rad1 mutant strains. Similar to previous studies, rad1 rad52 double mutants show a synergistic reduction in direct repeat recombination (35-fold). Interestingly, unrepaired heteroduplex is reduced 4-fold in the double mutants. Experiments with shortened repeats suggest that the reduction in unrepaired heteroduplex is due to decreased hDNA tract length in the double mutant strain.
SUP4异等位基因之间的直接重复重组检测可将未修复的异源双链DNA（hDNA）检测为扇形菌落。未修复异源双链的频率取决于错配情况，在产生C:C或G:G错配的构建体中最高，而在产生T:G或C:A错配的构建体中最低。此外，在pms1错配修复缺陷型菌株中，所有测试错配的未修复hDNA都会增加。这些结果支持了这样一种观点，即hDNA在重组事件中在SUP4重复序列之间形成，然后进行错配修复。研究了各种修复和重组缺陷突变对此检测的影响。未修复的异源双链仅在rad52突变株中显著增加。此外，rad52突变株中的直接重复重组减少了2倍，而在rad51、rad54、rad55和rad57突变体中，直接重复重组增加了3至4倍。切除修复基因RAD1中的突变不影响直接重复重组的频率。然而，rad1突变株中未修复异源双链的水平略有下降。与先前的研究相似，rad1 rad52双突变体在直接重复重组中表现出协同降低（35倍）。有趣的是，双突变体中未修复的异源双链减少了4倍。对缩短重复序列的实验表明，未修复异源双链的减少是由于双突变株中hDNA片段长度的减少。
